PROJECT 3 This Project will study the actions of steroid anesthetics on transmitter-gated ion channels. The focus is on potentiation of responses of the GABA-A receptor by steroids, as potentiation is most clearly related to anesthetic actions. A central goal of the work is to define the structural requirements for specific steroid effects. Work in the present funding period has shown that steroids have 3 distinct kinetic effects on the GABA-A receptor, and that the effects are mediated by binding to at least 2 sites. We will use biophysical analyses of evoked currents to determine the structures of the steroid molecule which are required for these effects, in complementary experiments, we will study the consequences of mutations to proposed steroid binding regions in the GABA-A receptor. We will also perform experiments to identify the location of the steroid binding pocket in the receptor with respect to the cell membrane, that is, whether it is located in the inner or outer leaflet of the membrane. In studies performed during the present funding period, we have found that the effects of steroids depend on the concentration of transmitter (GABA) used to activate the receptor, which may have a significant role in shaping the physiological actions of steroids. We will study the basis for the this relationship. We will continue to produce recombinant epitope tagged GABA-A receptors, for use in biochemical studies. Finally, we will determine the effects of steroids on the function of additional receptors, including the glycine a3 receptor. The proposed work builds on previous results from this Project and the Program as a whole. It is expected to lead to significant insights into the nature and location of the steroid binding sites on the GABA- A receptor. Further, it is expected to reveal the critical molecular mechanisms by which steroid anesthetics produce functional effects associated with anesthesia. Finally, it should assist in developing insights into the parts of the steroid molecule which are associated with specific functional consequences, which could result in increased specificity or efficacy for steroid anesthetics.